Enhanced maintenance of pregnancy using leukaemia inhibitory factor in embryo culturing

ABSTRACT

The present invention relates to the use of leukemia inhibitory factor (LIF) in the enhancement of development and maintenance of mammalian embryos, particularly sheep embryos. It has been observed that following the introduction into foster mothers of embryos cultured in vitro in the presence of LIF, the maintenance of pregnancy is enhanced relative to that seen following introduction of embryos that had not been cultured with LIF prior to introduction into foster mothers.

The present invention relates to the use of Leukemia Inhibitory Factor(LIF) in the enhancement of development and maintenance of animal ormammalian embryos and the use of same to enhance impregnation.

LIF is a protein that has previously been cloned, produced and purifiedin large quantities in recombinant form from both E. coli and yeastcells (International patent application Ser. No. PCT/AU88/00093). LIFhas been defined as a factor, the properties of which include:

1. the ability to suppress the proliferation of myeloid leukaemic cellssuch as M1 cells, with associated differentiation of the leukaemiccells; and

2. the ability to compete with a molecule having the defined sequence ofmurine LIF or human LIF (defined in International patent applicationSer. No. PCT/AU88/00093) for binding to specific cellular receptors onM1 cells or murine or human macrophages.

A major difficulty associated with present in vitro fertilisation (IVF)and embryo transfer (ET) programs, particularly in humans, is the towsuccess rate "achieved" on implantation of fertilised embryos.Currently, in human IVF programs, the implantation rate may be as low as10%, leading to the present practice of using up to four fertilisedembryos in each treatment which, in turn, leads occasionally to multiplebirths. Accordingly, there is a need to improve the implantation rate inhuman IVF programs. Similarly, in IVF and ET treatments in domesticanimals such as sheep, cattle, pigs and goats, it is highly desirablefor economic reasons to have as high an implantation rate as possible soas to reduce the numbers of fertilised embryos lost and unsuccessfultreatment procedures performed. Furthermore, as with human IVFprocedures, the practice of transferring more than one embryo to therecipient animal to ensure pregnancy can result in unwanted multiplebirths.

One major constraint with embryo transfer is the need to hold embryos inculture media for either relatively short periods of time, perhaps onlya few hours prior to transfer or for longer periods of some days, aftermicromanipulation.

In the development of a mammalian embryo, the fertilised egg passesthrough a number of stages including the morula and the blastocyststages. In the blastocyst stage, the cells form an outer cell layerknown as the trophectoderm (which is the precursor of the placenta) aswell as an inner cell mass (from which the whole of the embryo proper isderived). The blastocyst is surrounded by the zona pellucida, which issubsequently lost when the blastocyst "hatches". The cells of thetrophectoderm are then able to come into close contact with the wall ofthe uterus in the implantation stage. Prior to formation of the embryoproper by the inner cell mass by gastrulation, the whole cell mass maybe referred to as "pre-embryo".

Embryo mortality has been attributed to incomplete hatching of theblastocyst from the zona pellucida and/or unsuccessful implantation ofthe embryo to the uterine wall, possibly due to spontaneousdifferentiation of the embryonic stem cells (ES) during their period inculture prior to transplantation.

In accordance with the present invention, it has been found that whenLIF is included in an in vitro embryo culture medium, the hatchingprocess is enhanced leading to an increased number of embryos completingthe development stage by undergoing developmental changes associatedwith implantation. Thus, LIF is an embryo protective agent. As a result,the implantation rates for IVF and ET programs can be, and are,significantly improved by the use of LIF in the in vitro embryo culturemedium.

Furthermore, media containing LIF is suitable for use in earlymanipulative procedures on the oocyte/embryo such as in vitrofertilisation, embryo splitting and nuclear transfer where survivalrates of embryos are low. LIF also has important applications in thegrowth of totipotent stem cell lines for cloning for inclusion into themedia used for the transport of cooled or frozen embryos/semen.

Unless otherwise specified, use of "LIP" herein refers to syntheticrecombinant or naturally occurring human, murine and/or livestock orruminant LIF, such as from sheep, pigs, cows, goats, donkeys and horsesand from other animals such as dogs, cats or birds (eg. chickens) and toderivatives or parts thereof. Such derivatives or parts thereof includeany one or more contiguous series of amino acids contained within anyone of the above LIEF molecules and includes single or multiple aminoacid substitutions, deletions and/or additions to or in the natural orsynthetic LIF molecule. Conditions for preparing recombinant LIF aredisclosed in International Patent Application Nos PCT/AU88/00093 andPCT/AU90/00001 although variations and/or modifications to theseconditions may vary depending on the host cell used. Any such variationsand/or modifications are within the scope of the subject invention. Thehost cells may be eukaryotic (eg. yeast, mammalian, insect, plant etc.)or prokaryotic (eg. Escherichia coli, Bacillus sp, Pseudomonas sp etc.)cells.

Accordingly, one aspect of the present invention contemplates a methodfor enhancing the impregnation rate in an animal with one or moreembryos said method comprising maintaining and/or developing the embryosin a medium containing an effective amount of leukaemia inhibitoryfactor (LIF) for sufficient time and under appropriate conditions andthen implanting the embryos into the animal.

By "impregnation" means the rate of successful implantations andsubsequent development of a fertilised embryo in

Another aspect of the present invention contemplates a method formaintaining embryos or pre-embryos in in ELtX culture while retainingviability for use in embryo transfer, IVY: and/or genetic manipulationwhich method comprises culturing said embryos in a medium containing aneffective amount of LIF for sufficient time and under appropriateconditions.

This method of maintaining the viability of embryos in culture haspotential for allowing genetic manipulation of the whole embryo. Suchsuccessful genetic manipulation is restricted at the present time due tothe limited amount of time available to perform experiments on viableembryos.

The method also may be advantageous in maintaining viability of embryosunder transport conditions and may also be beneficial in the storage ofembryos when compared to techniques currently employed.

Another aspect of the present invention relates to a method forenhancing the in vitro development of a mammalian embryo to theimplantation stage, which method comprises the step of culturing theembryo in vitro in a culture medium containing an effective amount ofmammalian LIF.

As is demonstrated below the inclusion of LIF in the culture mediumprior to the formation of the blastocyst, or both prior to and followingblastocyst formation, also increases the number of pre-embryoscompleting the developmental stage by undergoing development changesassociated with implantation. The addition of LIF also reduces thenumber of pre-embryos degenerating while in culture. As a result, theimplantation rate for IVF and ET programs can be significantly improvedby use of LIF in the in vitro culture medium.

"Animal embryos" is used in its broadest sense encompassing embryos frommammals such as humans, ruminant and other livestock animals and otheranimals such as birds and fish. It will be appreciated that while thesubject invention is exemplified herein by the development ovine embryosin vitro, the present invention extends to the use of LIF in thedevelopment of embryos of other animal or mammalian species includinghumans, ruminants and animals such as cattle, horses, donkeys, goats andthe like.

The present invention, also extends to a method for in vitrofertilisation and subsequent implantation of a mammalian embryo which ischaracterised in that the embryo is cultured in vitro in a culturemedium containing an effective amount of mammalian LIF prior to transferinto animal or mammalian host, where "host" is defined as a suitablyreceptive female animal or mammal.

A further aspect of the present invention relates to a non-human animaland in particular a chimaeric non-human animal or transgenic progeny ofsaid animal generated by known techniques using ES cells which have beenmaintained in vitro in LIF-containing culture medium. In accordance withthis aspect of the present invention, ES cells are derived from animalembryos passaged in a culture medium containing LIF wherein said EScells have additional genetic material inserted therein. The transgenicanimals contemplated include nonhuman mammals such as livestock andruminant animals and domestic animals.

In accordance with the present invention, "homologous" or "heterologous"systems may be employed meaning that the animal from which LIF isderived is the same animal (homologous) or a different animal(heterologous) from which the embryos are isolated. The LIF may benaturally occurring but is preferably recombinant or synthetic LIF. TheLIF may be of any origin such as human, murine or livestock animal(including ruminant animal) LIF provided that the LIF has the desiredeffect. It may be, for example, that a LIF from one animal may not be asactive as a LIF from another animal. It is within the skill of theaddressee to readily screen for suitable LIFs from appropriate animals.Where recombinant LIF is used, it may be produced in eukaryotic orprokaryotic cells.

The present invention is also directed to composition comprising aneffective amount of LIF in combination with an animal (eg. mammalian)embryo maintaining medium. The present invention also provides acomposition having embryotrophic and/or embryo protective propertiescomprising LIF in combination with one or more of ovine trophoblastprotein, Interleukin 1 and/or 2, macrophage colony stimulating factors,platelet activating factor, a factor in the murine fibroblast 3T 3 lineand/or plasminogen. The above composition may also be in combinationwith an embryo maintaining medium.

An embryo maintaining medium as contemplated herein includes but is notnecessarily limited to SOF and/or M2.

The amount of LIF used in accordance with the present invention is thatrequired to maintain and/or develop embryos and/or enhance impregnationand is in the range of 100 units/ml to 10,000 units/ml, preferably 500units/ml to 5,000 units/ml and most preferably from 1,000 units/ml to5,000 units/mi. A unit of LIF activity is defined in PCT/AU88/00093.

The present non-limiting examples further illustrate the presentinvention:

EXAMPLE 1 Materials and Methods EMBRYO COLLECTION

Forty mature Merino ewes were treated for 14 days with a CIDR containing0.3 g progesterone (Riverina Artificial Breeders, NSW) and weresuperovulated by the injection (i.m.) of 5 mg of the pituitaryfollicle-stimulating hormone FSH-P (Intervet Australia Ply, Ltd, Sydney,NSW) and 200 iu of PMSG (i.m.) (Pregnecol; Heriot Agvet Pty, Ltd,Melbourne, Vic) 48 h before CIDR withdrawal, followed by decreasingdoses of FSH-P (4, 3, 3, 2, and 1 mg) every 12 h afterwards. Ovulationwas ensured by a 50 μg injection (i.m.) of Gonadotrophin-Releasinghormone (GnRH;Auspep Pry, Ltd, Melbourne, Vic) 24 h after CIDRwithdrawal, which coincided with the time that most ewes were detectedin oestrus by vasectomised rams fitted with marking crayons. Ewes wereinseminated via a laparoscope with approximately 100×10⁶ fresh motilesperm into each uterine horn 8-12 h after oestrus. The semen wascollected from 4 fertile rams, diluted 1:1 in phosphate buffered saline(Flow Laboratories, North Ryde, NSW) and kept at ambient temperatureprior to insemination. Six days after oestrus the ewes wereanaesthetised with an injection (i.v.) of the barbiturate sodiumthiopentone (Intraval: May & Baker, Footscray, Vic) and maintained undergeneral anaesthesia by a mixture of halothane (May & Baker) and oxygen.The uterus was exteriorised by a midventral laparotomy and each horn wascannulated with a Foley catheter (Promedica, Moorabin, Vic) at approx. 1cm below the bifurcation. A 20 g needle attached to a 20 ml syringefilled with flushing media was passed into the lumen of the uterus nearthe uterotubal junction. Each horn of the uterus was flushed and themedia plus embryos were collected into glass vessels via a Foleycatheter. Embryos were pooled in M2 holding media containing 4 mg/mlMiles BSA (Pentex Crystalline; Miles Diagnostics, Kanakee, Ill., USA)and graded according to health and stage of development beforeallocation to treatment groups.

CULTURE OF EMBRYOS

Embryos (166 assessed as healthy and 17 as poor quality) were randomlyallocated to one of three treatment groups. Embryos in treatment 1 (80healthy and 5 poor quality embryos at the morula or early blastocyststage) were cultured in lots of 10 for 1-2 h in 5 ml of M2 holding mediaprior to being transferred either as pairs (Group 1; n=38) or singularly(Group 2; n=47) into recipient ewes at Day 6 of the oestrous cycle.Group 3 embryos (44 healthy and 6 poor quality embryos at the morula orearly blastocyst stage), were cultured individually in SOF media for 48h prior to transfer. Three separate 100 μl droplets of SOF culture mediawere placed in a 35×10 mm plastic Petri-dish and covered with 2.5 mlparaffin oil (Labchem, Ajax Chemicals, Auburn, NSW). One embryo was thenplaced in each droplet and the Petri-dishes were placed in awaterjacketed incubator and were maintained at 39° C. in an atmospherecontaining 20%O₂, 5% CO₂ and 75% N₂. Group 4 embryos (44 healthy and 6poor quality morula or early blastocysts) were cultured for 48 h in SOFculture media as for Group 3 except that human recombinant LIF was addedto the media at a rate of 1000 units/ml).

The number of healthy or poor quality (degenerating) embryos, thoseachieving morula, blastocyst or hatching blastocyst stage of developmentwas recorded at the time of embryo collection, every 12 h afterwards andimmediately prior to transfer to recipient ewes at Day 8 after oestrus.

CULTURE MEDIA

The flushing media used was Dulbecco's phosphate buffered salinecontaining 10% (v/v) foetal calf serum, Penicillin G. potassium salt(0.060g/l) and Streptomycin sulphate (0.050g/l).

M2 culture media (Quinn et al, 1982) was a modified Krebs-Ringersolution with some of the bicarbonate substituted with HEPES buffer. Thecomponents were HEPES buffer (4.969 g/l), NaCl (5.533 g/l), KCl (0.356g/l), CaCl₂.2H₂ O (0.252 g/l), KH₂ PO₄ (0.162 g/l), MgSO₄.7H₂ O (0.293g/l), NaHCO₃ (0.349 g/l, Penicillin G. potassium salt (0.060 g/l),Streptomycin sulphate (0.050 g/l) made up to 1 liter with Millipore H₂O.

SOF culture media (Tervit et al., 1972) consisted of NaCl (6.95 g/l),KCl (0.534 g/l), KH₂ PO₄ (0.162 g/l), CaCl₂ 2H₂ O (0.252 g/l), MgCl₂ 6H₂O (0.10 g/l), NaHCO₃, (2.106 g/l), Na lactate (0.616 g/l), Na pyruvate(0.0363 g/l), Glucose (0.270 g/l), BSA (32.0 g/l), Penicillin G.potassium salt (0.060 g/l), Streptomycin sulphate (0.050 g/l) made up to1 litre with Millipore H₂ O.

All media was sterilised by filtration through a 0.2 μm filter(Millipore Pry, Ltd, Richmond, Vic).

EMBRYO TRANSFER

Two hundred 3 year old malden Merino ewes had their oestrous cyclessynchronised with a 14 day CIDR treatment. An injection of 400 UI PMSGwas given at the time of CIDR withdrawal. Vasectomised rams, fitted withharnesses and crayons were placed with the ewes to detect oestrus. Ewesobserved in oestrus were randomly allocated to one of 4 recipientgroups. Recipient ewes were treated with local anaesthetic (lignocaine,Lyppard Chemicals, Brighton, Vic) and using laparoscopy, the number ofcorpora lutea on each ovary was recorded. The uterus was located and thetip of the uterine horn, ipsilateral to an ovulating ovary wasexteriorised through a 2 cm midventral incision. The uterus waspunctured approximately 4 cm from the tip and the embryo, bathed in Msholding media was deposited via a Tomcat catheter (Size 3.5 FR, LyppardChemicals, Brighton, Vic) attached to a 1 ml syringe. The uterine hornwas returned into the abdominal cavity and the incision sutured.

All recipient ewes were grazed with harnessed vasectomised rams for 21days to provide an estimate of embryonic loss prior to or atimplantation (Edey, 1967). Ewes were scanned using ultrasonics on Days70 of pregnancy to determine the number of healthy foetuses.

ANALYSIS

Differences between groups were determined by Chi-square analysis.

EXAMPLE 2 Effect Of LIF On In Vitro Development Of Ovine Embryos To ThePost-Hatching Stage EMBRYO CULTURE

The addition of 1,000 units/ml of human recombinant LIF to SOF culturemedia significantly improved the development (more blastocysts hatchingand less degenerating) of healthy morula and blastocyst embryos culturedfor 48 h in vitro (Table 1 ).

Both treatment groups had 6 embryos initially classified as poor at thetime of embryo recovery. Over the treatment period their health did notimprove so they were discarded and not transferred to recipient ewes.

IMPLANTATION RATES

When the cultured embryos were individually transferred to recipientewes the 21-day non return rate (an indicator of implantation rate) ofewes receiving an embryo that had been cultured in SOF+LIF (Group 4) wassignificantly higher than that of ewes receiving an embryo culture inSOF alone (Group 3: Table 2). Furthermore, the implantation rate of theGroup 4 ewes was similar to recipient ewes that had received a singleembryo within 2 h of collection (group 2), but the non-return rates ofboth groups were lower than that of recipient ewes that had two embryostransferred soon after collection (Group 1).

Actual pregnancy rates, as determined by real time ultrasonic scanningon Day 70 of pregnancy are shown in Table 3.

The results given above demonstrate that the addition of humanrecombinant LIF to culture media increases up to 4-fold the number ofovine blastocysts that "hatch" from the zona pellucida and decrease thenumber of embryos degenerating during their 48 h culture in SOF at anatmosphere of 20% O₂, 5% CO₂ and 75% N₂. In fact 64% of the embryoscultured in SOF+LIF had hatched by Day 8 after oestrus which is similarto that found in vivo (Rowson and Moor, 1966, Bindon, 1971). LIF mayhave a role in maintaining the health of embryos under adverseconditions. Furthermore, during this period of culture the pregnancyrates of the recipient ewes after the transfer of embryos cultured inmedia containing LIF for 48 h was at least equal to that of ewesreceiving an embryo immediately after collection.

This stabilising role of LIF on embryos agrees with the recent reportsof the localisation of LIF receptors on the Day 4 murine embryo and ofmurine LIF being expressed strongly in the endometrial glands of the Day4 pregnant and pseudopregnant mouse. This suggests that LIF is producedas a passive response to pregnancy rather than to the presence of theembryo. LIF therefore appears to be produced at the time that the embryois entering the uterus from the oviduct. The results herein suggest thatsome of the embryonic mortality reported at around this time could beaverted by an adequate supply of LIF to the embryo. Currently, in mostembryo transfer programs in both humans and livestock animals, more thatone embryo is transferred to each recipient to ensure a viablepregnancy. Our study indicates that pregnancy rates are almost 40%higher when 2 embryos are transferred to each ewe even though there isstill about a 50% loss of embryos (52% of these ewes had twins, 37% hadsingles and 11% were not pregnant). Hence, there is a possibility thatLIF could prevent some of this embryonic loss and effectively make thetransfer of a single embryo to each recipient practical.

                  TABLE 1                                                         ______________________________________                                        Number and percentage of healthy embryos                                      hatching or degenerating during 48 h culture in media                         SOF (n = 42) or SOF + 1000 units/ml LIF (n = 44).                             Time    Hatched Embryos     Degenerating Embryos                              in Culture                                                                            SOF      SOF + LIF  SOF     SOF + LIF                                 ______________________________________                                         0 h    0      0%    0     0%   0    0%   0    0%                             12 h    0      0%    3     7%   2    5%   0    0%                             24 h    1      2%    10*  23%   2    5%   1    2%                             36 h    6     14%    19*  43%   5    11%  1    2%                             40-46 h 7     16%    28*  64%   13*  27%  4    9%                             ______________________________________                                         *Denotes significant differences between SOF & SOF + LIF treatment groups     P < 0.05.                                                                

                                      TABLE 2                                     __________________________________________________________________________    Ewes returning to service within 21 days after oestrus.                                   Group 1                                                                             Group 2                                                                             Group 3                                                                              Group 4                                                    1 Embryo                                                                            2 Embryos                                                                           1 Embryo                                                                             1 Embryo                                                   3 h in M2                                                                           3 h in M2                                                                           48 h in SOF                                                                          48 h in SOF + LIF                              __________________________________________________________________________    Returned to service                                                                        0    13    26     15                                             Not returned to service                                                                   19    28    18     27                                             Total       19    41    44     42                                             % Not Returned                                                                            100.sup.a                                                                           .sup. 68.sup.b                                                                      .sup. 41.sup.c                                                                       .sup. 64.sup.b                                 __________________________________________________________________________     Different superscripts denote significant differences (P < 0.05).        

                                      TABLE 3                                     __________________________________________________________________________    Number of recipient ewes pregnant at Day 70 after oestrus.                                Group 1                                                                             Group 2                                                                             Group 3                                                                              Group 4                                                    1 Embryo                                                                            2 Embryos                                                                           1 Embryo                                                                             1 Embryo                                                   3 h in M2                                                                           3 h in M2                                                                           48 h in SOF                                                                          48 h in SOF + LIF                              __________________________________________________________________________    Non-pregnant                                                                               2    20    37     21                                             Pregnant    17    22     7     21                                             Total       19    42    44     42                                             % Pregnant  .sup. 89.sup.a                                                                      .sup. 52.sup.b                                                                      .sup. 16.sup.c                                                                       .sup. 50.sup.b                                 __________________________________________________________________________     Different superscripts denote significant differences (P < 0.05).        

EXAMPLE 3 THE USE OF LIF IN EMBRYO CULTURE AND TRANSFER

Ovine embryos were collected from merino ewes 6 days after oestrus asdescribed above. This time, however, they were cultured individually for10 days in either SOF, SOF+murine LIF or SOF+human LIF and developmentassessed daily. The intention was to see if and how far the embryosdeveloped post hatching in media containing LIF such as whether theyreach the expanded blastocyst or trophoblast stages of development whichoccurs at around Days 10-12 in vivo. Secondly, the effect of murine LIFon the development of cultured sheep embryos was also investigated.

Embryos were collected from merino ewes in exactly the same manner as inExample 1. The only difference being that the ewes were superovulated byeither the FSH products Ovagen or RFSH-50 supplied by HorizonReproduction. The treatments were:

Group 1 (n=19); Embryos cultured individually in SOF alone.

Group 2 (n=18); Embryos cultured individually in SOF+murine LIF. mLIFadded at 3500 units/ml by mouse stem cell bioassay which equates toapprox. 8000 units/ml using the Ml cell bioassay.

Group 3 (n=20); Embryos cultured individually in SOF+human LIF (hLIF).hLIF added at 5000 units/ml by MI cell bioassay.

The culture conditions were identical to the above examples except thatembryo development was assessed daily rather than twice daily. Theatmospheric condition were as described above, i.e. 20% O₂, 5% CO₂, and75% N₂.

The results are shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Days in Culture                                                               Treatment                                                                           0  1   2   3   4   5   6   7   8   9   10                               __________________________________________________________________________    SOF   10 B                                                                             19 B                                                                              13 B                                                                              11 B                                                                               1 HB                                                                              1 HB                                                                             19 D                                                    9 M    6 D                                                                               8 D                                                                               9 B                                                                               5 B                                                                       9 D                                                                              13 D                                                 SOF+   9 B                                                                              4 M                                                                              18 B                                                                              15 B                                                                              14 B                                                                              11 B                                                                              20 D                                             mLIF   9 M                                                                             14 B     3 D                                                                               4 D                                                                               7 D                                                 SOF+  10 B                                                                              4 HB                                                                              4 FB                                                                             12 FB                                                                             14 FB                                                                             14 FB                                                                             14 FB                                                                             14 FB                                                                             12 FB                                                                             11 FB                                                                             20 D                             hLIF  10 M                                                                             16 B                                                                               9 HB                                                                              7 HB                                                                              4 HB                                                                              2 HB                                                                              1 B                                                                               1 B                                                                               8 D                                                                               9 D                                               5 B                                                                               2 B                                                                               1 B                                                                               1 B                                                                               5 D                                                                               5 D                                                                               8 D                                                                               9 D                                               2 D                                                                               2 D                                                                               3 D                                                                               3 D                                                 __________________________________________________________________________     Legend:                                                                       M = morula                                                                    B = blastocyst                                                                HB = hatching blastocyst                                                      FB = blastocysts free from the zona pellucida                                 D = degenerating embryos                                                 

After 3 days in culture significantly more embryos had developed tohatching or had completely hatched from the zona pellucida when placedin SOF+hLIF (Group 3) than those embryos in SOF (Group 1) or SOF+mLIF(Group 2) (P<0.01, Chi-square). Furthermore, significantly less haddegenerated after 6 days in culture (P<0.01, Chi-square). There was nosignificant effect of mLIF on the development or survival of the embryo.None of the blastocysts that had hatched from the zone pellucida hadcommenced to elongate prior to degeneration.

EXAMPLE 4

This example details a repeat of Example 2 but placing the embryos infresh media every second day in an attempt to induce further developmentof the hatched blastocyst.

The treatments were as follows and the results are shown in Table 5.

Treatments: Control - SOF

Group 1 - SOF+mouse LIF (5000 units/ml by Ml bioassay)

Group 2 - SOF+human LIF (5000 units/ml by Ml bioassay)

                  TABLE 5                                                         ______________________________________                                        Treat- Days in Culture                                                        ment   0      1      2    3    4    5    6    7    8                          ______________________________________                                        SOF    2 B    2 B    2 B  8 D                                                 n = 8  6 M    6 M    5 M                                                                           1 D                                                      SOF+   3 B    8 B    1 HB 8 D                                                 mLIF   5 M           5 D                                                      n = 8                3 D                                                      SOF+   3 B    1 HB   2 FB 5 FB 6 FB 6 FB 6 FB 8 D                             hLIF   5 M    5 B    3 HB 1 HB 2 D  2 D  2 D                                  N = 8         2 M    1 B  2 D                                                                      1 M                                                                           1 D                                                      ______________________________________                                    

This experiment again demonstrates that hLIF improved the health ofmorula and blastocysts held in culture. While the hatched blastocystsgrew from about 1 mm to 2 mm they did not commence to elongate.

EXAMPLE 5

The aim of this example was to investigate if the addition of LIF to SOFculture media improves the health of very early stage embryos.

In this experiment, embryos were taken from donor ewes at various timesafter oestrus and cultured for specific periods of time in SOF orSOF+hLIF (1000 units/ml) before their development was assessed bycounting their number of cells. All embryos (excepting those of group 5)were cultured in atmospheric conditions of low O₂ tension (7%) to allowthe earliest stage embryos to pass through the 8/16 cell block. Embryohealth was assessed by spreading the cells on a slide and ciunying cellnumbers.

Treatments:

Group 1. 2-4 cell embryos collected 2 days after oestrus and culturedfor 6 days in either SOF (n=10) or SOF+hLIF (n=14).

Group 2. 2-4 cell embryos collected 2 days after oestrus and culturedfor 2.5 days in either SOF (n=15) or SOF+hLIF (n=18).

Group 3. 8/16 cell embryos collected 4 days after oestrus and culturedfor 2 days in either SOF (n=11 ) or SOF+hLIF (n=9).

Group 4. Morula/blastocysts collected 6 days after oestrus and culturedfor 2 days in either SOF (n=19) or SOF+hLIF (n=20).

All these embryos were cultured in the specific atmosphere of 7% O₂, 5%CO₂, 88% N₂.

Group 5. Morula/blastocysts collected 6 days after oestrus and culturedfor 2 days in either SOF (n=19) or SOF+hLIF (n=18).

These embryos were cultured in an atmosphere of 20% O₂, 5%CO₂, 75% N₂.In other words this group is a repeat of the embryos cultured inExamples 2, 3 and 4.

The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                  Mean cell number (± sem)                                         Treatment SOF               SOF + hLIF                                        ______________________________________                                        Group 1   41.2 ± 3.94    69.1* ± 8.42                                   Group 2    7.4 ± 0.52     8.5 ± 0.60                                    Group 3   17.7 ± 1.94    24.9 ± 5.20                                    Group 4    75.7 ± 10.40   82.7 ± 11.49                                  Group 5   56.2 ± 5.90    62.6* ± 8.42                                   ______________________________________                                         *Denotes significant differences between groups within rows (P < 0.05)        Students ttest.                                                          

In summary, this experiment shows that the addition of hLIF to theculture media improves the health when they are held under sub-optimalconditions i.e. less than ideal atmospheric conditions (Group 5) or forlong periods of time under improved atmospheric conditions (Group 1).

EXAMPLE 6

The aim of this example was to determine if the addition of hLIF to thetransfer media improves the subsequence implantation rates of recipientewes.

In this experiment embryos were collected from superovulated Merino ewes6 days after oestrus. They were placed in either SOF or SOF+hLIF (5000units/ml) and individually intransferred to recipient ewes either within1 h of collection or after being held for between 6-8 h in the media at39° C. The embryos were transferred in the culture media. The pregnancyrates were confirmed by ultrasonic scanning 65 days after oestrus.

The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                         Time                                                         Group Media      in culture Pregnant                                                                              Not Pregnant                              ______________________________________                                        1     SOF        <1 h       21 (58%.sup.a)                                                                        15                                        2     SOF + LIF  <1 h       21 (51%.sup.a)                                                                        20                                        3     SOF        6-8 h       8 (21%.sup.b)                                                                        30                                        4     SOF + LIF  6-8 h      21 (53%.sup.a)                                                                        19                                        ______________________________________                                         Different superscripts denote significant differences (P < 0.01;              Chisquare)                                                               

This experiment demonstrates that hLIF has little effect uponimplantation rates if the embryos are transferred immediately aftercollection. However, if the embryos are left in culture for between 6-8h prior to transfer, which quite often is the situation in the field,the addition of hLIF to the media maintains the viability embryos whichwould otherwise have degenerated.

EXAMPLE 7

In a commercial ET program, 248 recipient Merino or Crossbred ewes eachreceived 2 embryos that had been held in either M2 or M2+hLIF (5000units/ml). The culture time varied from about 1 to about 6-8 hours. Thedonor ewes were Merinos.

The results are shown in Table 8.

                                      TABLE 8                                     __________________________________________________________________________           N   2 Embryos                                                                              1 Embryo Not Pregnant                                     __________________________________________________________________________    M2                                                                            Merinos                                                                              148 44       40       64                                               Xbred   20 10        4        6                                               Total  168     .sup. 54 (32%)                                                                         .sup. 44 (26%)                                                                         .sup. 70 (42%)                               M2 + LIF                                                                      Merinos                                                                               56 24       12       20                                               Xbred   44 24        7       13                                               Total  100     .sup. 48 (48%)                                                                         .sup. 19 (19%)                                                                         .sup. 33 (33%)                               __________________________________________________________________________     Embryo Mortality:                                                             M2 = 55%                                                                      M2 + LIF = 43%                                                           

The addition of LIF to the M2 culture media significantly increased theimplantation rates (P<0.05: Chi-square). This indicates that LIF haspotential as an embryo protective agent when added to media used for ETprograms.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

Bindon, B. M. (1971) Systematic study of preimplantation stages ofpregnancy in the sheep. Aust. J. biol. Sci. 24: 131-147.

Edey, (1967) J. Reprod. Fertil. 13: 437-443.

Quinn, P., Barros, C. and Whittington, D. G. (1982) Preservation ofhamster oocytes to assay the fertilising capacity of human spermatozoaJ. Reprod. Fert. 66: 161-168.

Rowson, L. E. A. and Moor, R. M. (1966) Development of the sheepconceptus during the first fourteen days. J. Anat. 100: 777-785.

Tervit, H. R., Whittington, D. G. and Rowson, L. E. A. (1972) Successfulculture in vitro of sheep and cattle ova. J. Reprod. Fert. 30: 493-496.

We claim:
 1. A method of enhancing the maintenance of pregnancy in amammal into which an embryo has been introduced, wherein said mammal isselected from the group consisting of ruminants, humans, pigs, donkeys,horses, dogs and cats, said method comprising prior to saidintroducing,culturing at least one embryo in a medium containing anamount of leukemia inhibitory factor for sufficient time and underappropriate conditions so as to effect an enhancement of the maintenanceof pregnancy in said mammal.
 2. The method according to claim 1 whereinsaid mammal is selected from the group consisting of human, sheep, pig,cow, goat, donkey, horse, dog and cat.
 3. The method of claim 2 whereinsaid mammal is a sheep.
 4. The method according to claim 1 wherein saidLIF is of human or murine origin.
 5. The method according to claim 1wherein said LIF is of sheep, pig, cow, goat, donkey, horse, dog or catorigin.
 6. The method according to claim 1, wherein the effective amountof LIF is from about 100 units/ml to about 10,000 units/ml.
 7. Themethod according to claim 6 wherein the effective amount of LIF is fromabout 500 units/ml to about 5,000 units/ml.
 8. The method according toclaim 7 wherein the effective amount of LIF is from about 1000 units/mlto about 5,000 units/ml.
 9. The method according to claim 1 wherein themedium for maintenance of the embryo is SOF or M2 medium.
 10. The methodof claim 1 wherein said mammal is a ruminant.
 11. The method of claim 10wherein said ruminant is a sheep.